Chemo-nociception is the process whereby primary afferent sensory neurons recognize and respond to noxious substances, resulting in pain, discomfort, and/or neurogenic inflammation. Such substances include exogenous environmental irritants, as well as endogenous pro-algesic agents that are produced or released in response to injury or disease. The ion channel TRPA1 also known as the 'wasabi receptor' functions as a major detector of chemical irritants and contributes significantly to mechanisms underlying acute pain and chronic inflammatory pain syndromes, such as arthritis, inflammatory bowel disease, hemorrhagic cystitis, asthma and other constrictive airway disorders. Thus, understanding basic mechanisms whereby TRPA1 detects noxious chemical stimuli and responds to cellular regulatory mechanisms is relevant to both basic sensory physiology and pain therapeutics. Recent studies have shown that chemically reactive (electrophilic) irritants activate TRPA1 through an unusual process involving covalent modification of cysteine residues within the so-called 'linker region' located in the cytoplasmic amino-terminus of the channel protein. An ankyrin-repeat rich domain (ARD) adjacent to the linker region also plays a significant role in specifying stimulus sensitivity, suggesting that it engages in a functional, and possibly structura interaction with the linker region to mediate irritant-evoked channel gating. Intracellular calcium also serves as a key modulator of TRPA1 function, promoting both potentiation and inactivation. Recent structure-function studies further suggest that calcium-dependent modulation is also specified by a site(s) within the ARD, although the cellular and biochemical mechanisms underlying this important regulatory process remain obscure. G protein beta-gamma subunits may also serve as TRPA1 modulators. The goal of this proposal is to elucidate biochemical and structural mechanisms whereby the TRPA1 amino terminus functions as an integrator of physiological stimuli that regulate sensory neuron excitation. The specific aims are to: (i) define the key structural elements of the linker region - such as length, proximity to the ARD domain, and relative location of modifiable cysteine residues that specify irritant detection and efficienc of channel gating; (ii) elucidate cellular signaling mechanisms underlying G protein and calcium-dependent channel modulation using electrophysiological and biochemical methods; and (iii) develop a structural model of the TRPA1 amino-terminus using biochemical and crystallographic methods. Together, these studies will provide a rational basis for the development and design of novel analgesic agents for controlling chronic pain and neurogenic inflammatory syndromes. PUBLIC HEALTH RELEVANCE: This project is focused on delineating basic cellular mechanisms that contribute to pain sensation. Results from these studies will enhance our understanding of persistent pain syndromes and help to identify therapeutic strategies (such as novel drugs) for treating chronic pain and associated inflammatory disorders.